Jim Houston writes- >The optimum excitation of 33342 is with the 351 line of the argon laser. >With emmision around 460+/-. >When using the 325 line of the HeCd can I expected a lower emmision output? Excitation at 325 mW gives about 56% of the emission intensity you'd get with equivalent power at 350 nm. >I would like to run the SP population protocol that was published by Stroms >et al. in Blood 15 September 2000-Vol 96. >They indicated using the 351 line in combination with a 450DF20 filter for >the Blue and a LP675 for the Red. > >The Question??? Will the 450/20 work with the 325 line or do I need expect >it to shift down.?? The 450/20 line is fine; the primary emission does not shift down with excitation wavelength. However, one might expect relatively more or less excitation of the red fluorescence, depending on the chemistry involved. The primary or orthochromatic (green) emission of single molecules acridine orange has an excitation maximum near 488 nm, but, while the red metachromatic fluorescence of oligomolecular complexes is adequately excited at 488 nm, its excitation maximum is at a shorter wavelength (around 460 nm, if I remember correctly). I don't recall reading anything about the spectral details of the far red emission from Hoechst, but I wouldn't be surprised if its excitation maximum was shifted away from 350 nm, probably down (in which case you'd actually gain something by using the 325 nm laser), but possibly up (in which case you'd lose). -Howard
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